The hagfish genome and the evolution of vertebrates.

As the only surviving lineages of jawless fishes, hagfishes and lampreys provide a crucial window into early vertebrate evolution1-3. Here we investigate the complex history, timing and functional role of genome-wide duplications4-7 and programmed DNA elimination8,9 in vertebrates in the light of a chromosome-scale genome sequence for the brown hagfish Eptatretus atami. Combining evidence from syntenic and phylogenetic analyses, we establish a comprehensive picture of vertebrate genome evolution, including an auto-tetraploidization (1RV) that predates the early Cambrian cyclostome-gnathostome split, followed by a mid-late Cambrian allo-tetraploidization (2RJV) in gnathostomes and a prolonged Cambrian-Ordovician hexaploidization (2RCY) in cyclostomes. Subsequently, hagfishes underwent extensive genomic changes, with chromosomal fusions accompanied by the loss of genes that are essential for organ systems (for example, genes involved in the development of eyes and in the proliferation of osteoclasts); these changes account, in part, for the simplification of the hagfish body plan1,2. Finally, we characterize programmed DNA elimination in hagfish, identifying protein-coding genes and repetitive elements that are deleted from somatic cell lineages during early development. The elimination of these germline-specific genes provides a mechanism for resolving genetic conflict between soma and germline by repressing germline and pluripotency functions, paralleling findings in lampreys10,11. Reconstruction of the early genomic history of vertebrates provides a framework for further investigations of the evolution of cyclostomes and jawed vertebrates.

The hagfish genome and the evolution of vertebrates.

As the only surviving lineages of jawless fishes, hagfishes and lampreys provide a critical window into early vertebrate evolution. Here, we investigate the complex history, timing, and functional role of genome-wide duplications in vertebrates in the light of a chromosome-scale genome of the brown hagfish Eptatretus atami. Using robust chromosome-scale (paralogon-based) phylogenetic methods, we confirm the monophyly of cyclostomes, document an auto-tetraploidization (1RV) that predated the origin of crown group vertebrates ~517 Mya, and establish the timing of subsequent independent duplications in the gnathostome and cyclostome lineages. Some 1RV gene duplications can be linked to key vertebrate innovations, suggesting that this early genomewide event contributed to the emergence of pan-vertebrate features such as neural crest. The hagfish karyotype is derived by numerous fusions relative to the ancestral cyclostome arrangement preserved by lampreys. These genomic changes were accompanied by the loss of genes essential for organ systems (eyes, osteoclast) that are absent in hagfish, accounting in part for the simplification of the hagfish body plan; other gene family expansions account for hagfishes' capacity to produce slime. Finally, we characterise programmed DNA elimination in somatic cells of hagfish, identifying protein-coding and repetitive elements that are deleted during development. As in lampreys, the elimination of these genes provides a mechanism for resolving genetic conflict between soma and germline by repressing germline/pluripotency functions. Reconstruction of the early genomic history of vertebrates provides a framework for further exploration of vertebrate novelties.

Sialome analysis of the cephalochordate Branchiostoma belcheri, a key organism for vertebrate evolution.

Sialic acid, a common terminal substitution of glycoconjugates, has been so far consistently identified in all vertebrates as well as in a growing number of bacterial species. It is assumed to be widely distributed among animal species of the deuterostome phylum, based on its identification in few echinoderm and all vertebrate species. However, whole sections of deuterostome, especially those intermediate species between invertebrates and vertebrates including cephalochordates, urochordates and hemichordates, are still unexplored in term of sialylation capacities. The discovery of functional sialic acid machinery in some of these species may shed new light onto the evolution of glycosylation capacities in deuterostome lineage. In a first approach, we investigated the sialylation pattern of a cephalocordate species, Branchiostoma belcheri, which occupies a strategic phylogenetic position to understand the transition of invertebrates toward vertebrates. Structural analysis of B. belcheri glycoconjugates established that this organism synthesizes large quantities of various sialic acids, some of which present rare or novel structures such as methylated sialic acids. These sialic acids were shown to be mainly associated with mono- and disialylated core 1-type O-glycans. Moreover, screening of the animal organs revealed the existence of exquisite tissue specificity in the distribution of sialic acids. Description of sialylation profiles was then correlated with the expression patterns of key enzymes involved in the biosynthesis of major forms of sialic acids, which provides the first complete overview of the sialylation patterns in cephalochordates.

Functional characteristics of amphioxus troponin in regulation of muscle contraction.

Troponin regulates contraction of vertebrate striated muscle in a Ca(2+)-dependent manner. More specifically, it acts as an inhibitor of actin-myosin interaction in the absence of Ca(2+) during contraction. In vertebrates, this regulatory mechanism is unlike that in some less highly derived taxa. Troponin in the smooth muscle of the protochordate ascidian species Halocynthia roretzi regulates actinmyosin contraction as an activator in the presence of Ca(2+), not as an inhibitor in the absence of Ca(2+) as is the case in vertebrates. In this study, contractile regulation of striated muscle from another protochordate, the amphioxus Branchiostoma belcheri, was analyzed using recombinant troponin components TnT, TnI, and TnC that were produced in an Escherichia coli expression system to further elucidate their roles in Ca(2+)-dependent regulation of the actin-myosin interaction. Combination of these troponin components in an actin-myosin ATPase activity assay showed that troponin in amphioxus striated muscle functions in a similar manner to troponin in vertebrate striated muscle, and differently from ascidian smooth muscle troponin. Thus, troponin function appears to have evolved differently in different protochordate muscles.

Molecular cloning and characterization of ligand- and species-specificity of amphibian estrogen receptors.

Estrogens are essential for normal reproductive activity in both males and females as well as for ovarian differentiation during a critical developmental stage in most vertebrates. To understand the molecular mechanisms of estrogen action and to evaluate estrogen receptor ligand interactions in amphibians, we isolated cDNAs encoding the estrogen receptors (ERalpha and ERbeta) from the Japanese firebelly newt (Cynops pyrrhogaster), Tokyo salamander (Hynobius tokyoensis), axolotl (Ambystoma mexicanum), and Raucous toad (Bufo rangeri). Full-length amphibian ER cDNAs were obtained using 5' and 3' rapid amplification of cDNA ends. The predicted amino acid sequences of these amphibian ERs showed a high degree of amino acid sequence identity (over 70%) to each other. We analyzed the relationships of these amphibian ER sequences to other vertebrate ER sequences by constructing a phylogenetic tree. We verified that these were bona fide estrogen receptors using receptor dependent reporter gene assays. We analyzed the effects of natural estrogens, ethinylestradiol, and DDT and its metabolites on the transactivation of the four amphibian species listed above, and Xenopus tropicalis ERs and found that there were species-specific differences in the sensitivity of these ERs to hormones and environmental chemicals. These findings will expand our knowledge of endocrine-disrupting events in amphibians.

A homolog of the vertebrate thyrostimulin glycoprotein hormone alpha subunit (GPA2) is expressed in Amphioxus neurons.

The cystine-knot glycoprotein hormone alpha (GPA) family regulates gonadal and thyroid functions in vertebrates. Little is known concerning GPA family members in primitive chordates. A previous genomic analysis revealed the presence of two genes homologous to the thyrostimulin alpha subunit (GPA2) in an amphioxus (Branchiostoma florideae); however only one GPA2 homolog contained both the cystine-knot structure and N-glycosylation site characteristic of family members. Gene-specific PCR was used to obtain the cDNA and genomic sequences of the GPA2 homolog of the amphioxus Branchiostoma belcheri. Whole-mount in situ hybridization revealed GPA2 mRNA expression in the anterior part of the nerve cord and on the left side of the central canal. Because amphioxus possesses only one true GPA2 homolog, while vertebrates possess two glycoprotein hormone alpha subunits (thyrostimulin alpha, or GPA2, and the common alpha subunit of gonadal and thyroid glycoprotein hormones, GPA1), our results suggest that GPA1 was acquired later in the vertebrate lineage through gene duplication.

Expression of the gene for ancestral glycoprotein hormone beta subunit in the nerve cord of amphioxus.

Amphioxus belongs to the subphylum cephalochordata, a clade of chordates phylogenetically placed at the most basal position. Despite many studies on the endocrine system of amphioxus, there were no confident lines of evidence on the presence of pituitary hormones, whereas recent amphioxus genome analysis reported that amphioxus has no pituitary hormone except for thyrostimulin, which is a glycoprotein hormone in the pituitary, brain, and other organs of vertebrates. In the present study, we cloned cDNA for one glycoprotein hormone beta subunit (GPB) from amphioxus, AmpGPB5, and phylogenetically indicated that AmpGPB5 is the ancestral molecule of glycoprotein hormone beta subunits of vertebrates including pituitary glycoprotein hormones. Synteny analyses showed conservation of chromosomal location of genes near GPB genes from amphioxus through human. The AmpGPB5 gene was expressed in a restricted region of the dorsal part of the nerve cord, glandular atrial cells of gills, and pre-vitellogenic oocytes in amphioxus. However, expression was not detected in the Hatschek's pit which is considered to be a primitive pituitary gland. On the basis of present results, we hypothesize that a portion of vertebrate pituitary hormones might be derived from an ancestral glycoprotein hormone of amphioxus that functions as a neuroendocrine hormone.

Evolution of cis-regulatory modules for the head organizer gene goosecoid in chordates: comparisons between Branchiostoma and Xenopus.

BACKGROUND: In cephalochordates (amphioxus), the notochord runs along the dorsal to the anterior tip of the body. In contrast, the vertebrate head is formed anterior to the notochord, as a result of head organizer formation in anterior mesoderm during early development. A key gene for the vertebrate head organizer, goosecoid (gsc), is broadly expressed in the dorsal mesoderm of amphioxus gastrula. Amphioxus gsc expression subsequently becomes restricted to the posterior notochord from the early neurula. This has prompted the hypothesis that a change in expression patterns of gsc led to development of the vertebrate head during chordate evolution. However, molecular mechanisms of head organizer evolution involving gsc have never been elucidated. RESULTS: To address this question, we compared cis-regulatory modules of vertebrate organizer genes between amphioxus, Branchiostoma japonicum, and frogs, Xenopus laevis and Xenopus tropicalis. Here we show conservation and diversification of gene regulatory mechanisms through cis-regulatory modules for gsc, lim1/lhx1, and chordin in Branchiostoma and Xenopus. Reporter analysis using Xenopus embryos demonstrates that activation of gsc by Nodal/FoxH1 signal through the 5' upstream region, that of lim1 by Nodal/FoxH1 signal through the first intron, and that of chordin by Lim1 through the second intron, are conserved between amphioxus and Xenopus. However, activation of gsc by Lim1 and Otx through the 5' upstream region in Xenopus are not conserved in amphioxus. Furthermore, the 5' region of amphioxus gsc recapitulated the amphioxus-like posterior mesoderm expression of the reporter gene in transgenic Xenopus embryos. CONCLUSIONS: On the basis of this study, we propose a model, in which the gsc gene acquired the cis-regulatory module bound with Lim1 and Otx at its 5' upstream region to be activated persistently in anterior mesoderm, in the vertebrate lineage. Because Gsc globally represses trunk (notochord) genes in the vertebrate head organizer, this cooption of gsc in vertebrates appears to have resulted in inhibition of trunk genes and acquisition of the head organizer and its derivative prechordal plate.

Platanus-allee is a de novo haplotype assembler enabling a comprehensive access to divergent heterozygous regions.

The ultimate goal for diploid genome determination is to completely decode homologous chromosomes independently, and several phasing programs from consensus sequences have been developed. These methods work well for lowly heterozygous genomes, but the manifold species have high heterozygosity. Additionally, there are highly divergent regions (HDRs), where the haplotype sequences differ considerably. Because HDRs are likely to direct various interesting biological phenomena, many genomic analysis targets fall within these regions. However, they cannot be accessed by existing phasing methods, and we have to adopt costly traditional methods. Here, we develop a de novo haplotype assembler, Platanus-allee ( http://platanus.bio.titech.ac.jp/platanus2 ), which initially constructs each haplotype sequence and then untangles the assembly graphs utilizing sequence links and synteny information. A comprehensive benchmark analysis reveals that Platanus-allee exhibits high recall and precision, particularly for HDRs. Using this approach, previously unknown HDRs are detected in the human genome, which may uncover novel aspects of genome variability.

Cephalochordate melanopsin: evolutionary linkage between invertebrate visual cells and vertebrate photosensitive retinal ganglion cells.

Animal photoreceptor cells can be classified into two distinct types, depending on whether the photopigment is borne on the membrane of a modified cilium (ciliary type) or apical microvilli (rhabdomeric type) [1]. Ciliary photoreceptors are well known as vertebrate rods and cones and are also found in several invertebrates. The rhabdomeric photoreceptor, in contrast, is a predominant type of invertebrate visual cell, but morphologically identifiable rhabdomeric photoreceptors have never been found in vertebrates. It is hypothesized that the rhabdomeric photoreceptor cell had evolved to be the photosensitive retinal ganglion cell for the vertebrate circadian photoentrainment [2, 3 and 4] owing to the fact that some molecules involved in cell differentiation are common among them [5]. We focused on the cephalochordate amphioxus because it is the closest living invertebrate to the vertebrates, and interestingly, it has rhabdomeric photoreceptor cells for putative nonvisual functions [6]. Here, we show that the amphioxus homolog of melanopsin [7, 8 and 9], the circadian photopigment in the photosensitive retinal ganglion cells of vertebrates, is expressed in the rhabdomeric photoreceptor cells of the amphioxus and that its biochemical and photochemical properties, not just its primary structure, are considerably similar to those of the visual rhodopsins in the rhabdomeric photoreceptor cells of higher invertebrates. The cephalochordate rhabdomeric photoreceptor represents an evolutionary link between the invertebrate visual photoreceptor and the vertebrate circadian photoreceptor.

Presence of sex steroids and cytochrome P450 genes in amphioxus.

The presence of sex steroids and their receptors has been demonstrated in all vertebrate groups from Agnatha to Mammalia but not in invertebrates. In genomic analyses of urochordates, cytochrome P450 (CYP) genes important for biosynthesis of sex steroids are absent. In the present study, we confirmed the presence of estrogen, androgen, and progesterone by using radioimmunoassay in gonads of amphioxus, Branchiostoma belcheri, which is considered to be evolutionarily closer to vertebrates than other invertebrates. Furthermore, CYP genes encoding CYP11A, CYP17, and CYP19 and transcripts for 17beta-hydroxysteroid dehydrogenase were cloned from amphioxus ovaries. Among invertebrates, the presence of hydroxysteroid dehydrogenase enzymes and metabolized steroids was shown in paracytic Taenia and corals. However, CYPs metabolizing sex steroids have not been demonstrated in invertebrates, nor has an attempt been made to consider the entire pathway from cholesterol to estrogen. This study is the first evidence to suggest the presence of CYP enzymes in sex steroid production in invertebrates.

Amphioxus homologs of Go-coupled rhodopsin and peropsin having 11-cis- and all-trans-retinals as their chromophores.

Because of low contents in the native organs and failure of the expression in cultured cells, the chromophore configurations of the pigments in Go-coupled opsin and peropsin groups in the opsin family are unknown. Here we have succeeded in expression of the amphioxus homologs of these groups in HEK293s cells and found that they can be regenerated with 11-cis- and all-trans-retinals, respectively. Light isomerized the chromophores of these opsins into the all-trans and 11-cis forms, respectively. The results strongly suggest that the physiological function of peropsin would be a retinal photoisomerase, while 11-cis configuration is necessary for the Go-coupled opsin groups.

Differentiation of Secondary Spermatogonia to Primary Spermatocytes by Mammalian Follicle-Stimulating Hormone in Organ Culture of Testes Fragments from the Newt, Cynops pyrrhogaster.

In order to elucidate essential factors responsible for the initiation and promotion of spermatogenesis, we developed an organ culture system with a chemically defined medium. When newt testes fragments, consisting of somatic cells and germ cells almost exclusively secondary spermatogonia, were cultured in control medium for three weeks, most of the testicular cysts still contained only secondary spermatogonia. On the other hand, in the medium supplemented with various kinds of hormones and vitamins primary spermatocytes (zygotene-pachytene) appeared in about 60% of the cysts by the second week. Selective removal of specific hormones and vitamins revealed that follicle-stimulating hormone (FSH) alone was indispensable and sufficient for the differentiation of secondary spermatogonia to primary spermatocytes. Neither the addition of luteinizing hormone (LH) nor androgens (testosterone and 5α-dihydrotestosterone) to the control medium stimulated differentiation. Consistent with these findings was the fact that radioreceptor assays revealed high affinity specific binding sites for FSH but none for LH. Since our ultrastructural studies revealed a major loss of contact between spermatogonia and Sertoli cells following exposure to FSH, we suggest that FSH triggers differentiation of spermatogonia by acting on Sertoli cells which in turn act on spermatogonia.

Gonadal state of wild amphioxus populations and spawning success in captive conditions during the breeding period in Japan.

Adult individuals of amphioxus (Branchiostoma belcheri) were collected by dredging from a research vessel at selected stations in two areas off the coast of Japan in July 2000: Deyama and Takamatsu so named by local fishermen in the Enshu Nada Sea. The number of males collected exceeded that of females at all the stations in Takamatsu and at four of five stations in Deyama. The over all sex ratio (males : females) of the collected animals was 1.2 : 1. The animals showed various maturational stages of the gonad, and approximately 70% had mature gonads. However, post-spawning animals were identified only at two stations in Takamatsu. Mature animals were placed in laboratory tanks. These animals remained in good conditions for about two months, and many animals spontaneously spawned in the tanks. This is the first report of spontaneous spawning of B. belcheri in Japan.

Acrosome reaction in spermatozoa from the amphioxus Branchiostoma belcheri (Cephalochordata, Chordata).

The formation of an acrosomal process at acrosomal exocytosis in spermatozoa of the amphioxus was described in the present report for the first time. A non-reacted acrosome was located in front of the nucleus, where a cup-shaped acrosomal vesicle covered a conical accumulation of subacrosomal material. When naturally spawned spermatozoa were treated with a calcium ionophore, ionomycin, the acrosomal vesicle opened at the apex and an acrosomal process was projected. The process exhibited a filamentous structure. The reaction followed the mode typically seen in marine invertebrates. These observations suggest that the features and function of the acrosome of amphioxus, whose position is on the border between invertebrates and vertebrates, reflect their ecological adaptation and phylogenic position.

Actin capping proteins, CapZ (ß-actinin) and tropomodulin in amphioxus striated muscle.

CapZ (ß-actinin) and tropomodulin (Tmod) are capping proteins involved in the maintenance of thin filaments in vertebrate skeletal muscles. In this study, we focused on amphioxus, the most primitive chordate. We searched for CapZ and Tmod genes in the amphioxus genome and determined their primary structures. Amphioxus possess one CapZα gene (CAPZA) and one CapZß gene (CAPZB), and the transcripts of these genes were found to be 67%-85% identical to those of human CapZ genes. On the other hand, amphioxus contain one Tmod gene (TMOD), and the product of this gene has an identity of approximately 50% with human Tmod genes 1-4. However, helix 2 of amphioxus Tmod, which is involved in protein-binding to tropomyosin, was highly conserved with approximately 74% identity to human Tmod genes. Western blotting indicated the presence of CapZ and Tmod in the striated muscle of amphioxus. These results suggest that unlike most of vertebrates, such as fish, amphibian, bird, and mammal, CapZ from amphioxus striated muscle is derived from two genes CAPZA and CAPZB, and Tmod is derived from one TMOD gene.

Amphioxus connectin exhibits merged structure as invertebrate connectin in I-band region and vertebrate connectin in A-band region.

Connectin is an elastic protein found in vertebrate striated muscle and in some invertebrates as connectin-like proteins. In this study, we determined the structure of the amphioxus connectin gene and analyzed its sequence based on its genomic information. Amphioxus is not a vertebrate but, phylogenetically, the lowest chordate. Analysis of gene structure revealed that the amphioxus gene is approximately 430 kb in length and consists of regions with exons of repeatedly aligned immunoglobulin (Ig) domains and regions with exons of fibronectin type 3 and Ig domain repeats. With regard to this sequence, although the region corresponding to the I-band is homologous to that of invertebrate connectin-like proteins and has an Ig-PEVK region similar to that of the Neanthes sp. 4000K protein, the region corresponding to the A-band has a super-repeat structure of Ig and fibronectin type 3 domains and a kinase domain near the C-terminus, which is similar to the structure of vertebrate connectin. These findings revealed that amphioxus connectin has the domain structure of invertebrate connectin-like proteins at its N-terminus and that of vertebrate connectin at its C-terminus. Thus, amphioxus connectin has a novel structure among known connectin-like proteins. This finding suggests that the formation and maintenance of the sarcomeric structure of amphioxus striated muscle are similar to those of vertebrates; however, its elasticity is different from that of vertebrates, being more similar to that of invertebrates.

Evolution of the reproductive endocrine system in chordates.

The cephalochordate, amphioxus, is phylogenetically placed at the most primitive position in the chordate clade. Despite many studies on the endocrine system of amphioxus, definitive evidence has not been reported for the presence an endocrine system comparable to the pituitary-gonadal axis, which is important in the regulation of reproduction in vertebrates. Recent genome analyses in the amphioxus, Branchiostoma floridae, showed that it does not have any pituitary hormone genes except the thyrostimulin gene. Thyrostimulin is a heterodimeric glycoprotein hormone consisting of α and ß subunits, and is present in various organs of vertebrates. Analyses of a phylogenetic tree and a synteny suggest that amphioxus' thyrostimulin is an ancestral type of the glycoprotein hormones in chordates. In addition, genes for sex steroidogenic enzymes belonging to the CYP family were found in the genome sequences. The conversion pathway of sex steroids from cholesterol to estrogen, androgen, and major sex steroids was also identified in the gonads of amphioxus in vitro. Furthermore, we demonstrated the expression of genes encoding thyrostimulin and sex steroidogenic enzymes by an in situ hybridization technique. Here, we discuss the evolution of hormones and reproductive functions in the neuroendocrine control system of chordates.

Estrogen-dependent transactivation of amphioxus steroid hormone receptor via both estrogen and androgen response elements.

Estrogens are necessary for ovarian differentiation during critical developmental windows in most vertebrates and promote the growth and differentiation of the adult female reproductive system. Estrogen actions are largely mediated through the estrogen receptors (ERs), which are ligand-activated transcription factors. To understand the molecular evolution of sex steroid hormone receptors, we isolated cDNAs encoding two steroid receptors from Japanese amphioxus, Branchiostoma belcheri: an ER ortholog and a ketosteroid receptor (SR) ortholog. Reporter gene assays revealed that the SR ortholog has molecular functions similar to those of the vertebrate ER. Surprisingly, the ER ortholog is an estrogen-insensitive repressor of SR-mediated transcription. Furthermore, we found that the SR ortholog can bind to both estrogen-responsive elements (EREs) and androgen-responsive elements (AREs) and mediates transcriptional activation by estrogens through both types of elements. Our findings suggest that the ancestral SR, but not ER, could bind estrone and induce the ERE- and ARE-dependent transactivation and that it gained the ability to be regulated by ketosteroid and recognize ARE specifically before jawless vertebrates split. These results highlight the importance of comparative experimental approaches for the evolutionary study of endocrine systems.